The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) has been implicated in the responses of plants to oxidative stress, suggesting a role for alternative polyadenylation. To better understand this, poly(A) site choice was studied in a mutant (oxt6) deficient in CPSF30 expression using a genomescale approach. The results indicate that poly(A) site choice in a large majority of Arabidopsis genes is altered in the oxt6 mutant. A number of poly(A) sites were identified that are seen only in the wild type or oxt6 mutant. Interestingly, putative polyadenylation signals associated with sites that are seen only in the oxt6 mutant are decidedly different from the canonical plant polyadenylation signal, lacking the characteristic A-rich near-upstream element (where AAUAAA can be found); this suggests that CPSF30 functions in the handling of the near-upstream element. The sets of genes that possess sites seen only in the wild type or mutant were enriched for those involved in stress and defense responses, a result consistent with the properties of the oxt6 mutant. Taken together, these studies provide new insights into the mechanisms and consequences of CPSF30-mediated alternative polyadenylation.

The ability of an ice-binding protein (IBP) from Marinomonas primoryensis (MpIBP) to influence ice crystal growth and structure in non-physiological pH environments was investigated in this work. The ability for MpIBP to retain ice interactivity under stressed environmental conditions was determined via (1) a modified splat assay to determine ice recrystallization inhibition (IRI) of polycrystalline ice and (2) nanoliter osmometry to evaluate the ability of MpIBP to dynamically shape the morphology of a single ice crystal. Circular dichroism (CD) was used to relate the IRI and DIS activity of MpIBP to secondary structure. Results illustrate that MpIBP secondary structure was stable between pH 6 -pH 10. It was found that MpIBP did not interact with ice at pH ≤ 4 or pH ≥ 13. At 6 ≤ pH ≥ 12 MpIBP exhibited a reduction in grain size of ice crystals compared to control solutions and demonstrated dynamic ice shaping at 6 ≤ pH ≥ 10. The results substantiate that MpIBP retains some secondary structure and function in non-neutral pH environments, thereby enabling its potential utility in non-physiological materials science and engineering applications.

No abstract

Modern high-throughput DNA sequencing has the potential to generate large volumes of data for analysis by investigators-including poly(A) site data. Here I describe a computational method to compare poly(A) site choice differences between two large data sets based on the relative abundance and position of tags within each reference sequence to which they are aligned. This method provides rapid quantification and visualization of differences and similarities in poly(A) site choice between the two datasets.

Synechococcus Sp. PCC 7002 is a rapidly growing, genetically tractable, photosynthetic microbe of industrial interest. Understanding how gene expression and carbon fixation are regulated in PCC 7002 is essential for its advancement as an industrial microbe. Towards that aim, we are investigating a cyanobacterial homolog to E. coli RNase II/R, A1543, that we hypothesize regulates gene expression and other cellular processes in response to changes in cellular redox state.A1543 is an RNA binding protein with three RNA binding domains ‐ two OB folds and an S1 domain. Multiple alignment of PCC 7002 A1543 to RNase II/R homologues in other cyanobacteria show conservation across the genre of two cysteines. Threading of the A1543 sequence onto the E. coli RNase II crystal structure shows that these two cysteines sit in L12 and b2 of the first OB fold, within 4 angstroms of each other, and thus could potentially interact via a disulfide bond.To characterize the role of A1543 in vivo we constructed a segregated A1543 knockout line, DA1543, which shows, as previously reported in the literature, defects in both pigmentation and growth. Additionally, we found that our DA1543 line shows resistance to the carbonic anhydrase acetazolamide compared to wild‐type. This phenotype had been characterized before in a related species, Synechocystis sp. 6803. We have found concentrations of acetazolamide that is lethal and inhibitory to both wildtype and DA1543 cells in several different growth conditions. Interestingly, at 3% CO2, acetazolamide has a greater effect on growth than in air for both cell lines.A1543 is a potential link between the cellular redox state and RNA processing in cyanobacterial cells via changes of RNA binding behavior through oxidation or reduction of a disulfide bridge in a conserved OB‐fold. Understanding if this type of regulation is occurring and how it affects cyanobacterial gene expression and carbon fixation will enable further development of Synechococcus Sp. PCC 7002 as an industrial research organism.Support or Funding InformationPatrick Thomas is supported by the NIH T32 Biophysics training grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.